We have developed a simple method to isolate myosin heavy chain (MHC) and actin from small human skeletal muscle samples for determination of their fractional synthesis rates, utilizing polyacrylamide gel electrophoresis (PAGE). The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects after they received a 14-hr intravenous infusion (prime = 7.58 (mol.kg-1, constant infusion = 7.58 (mol.kg-1.hr-1) of [1-13C]leucine. Overall, the synthesis rate of MHC was 20% lower (p=0.012) and the synthesis rate of actin was 61% higher (p=0.060, N.S.) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC/C/IRMS provides valuable information about the synthesis rates of these specific contractile proteins as opposed to the more general information provided by previous studies of MMP synthesis rates.